The proposed research in this continuation grant application is designed to evaluate the extent to which fluorescent probes of membrane potential, in conjunction with measurements of ionic fluxes, can be used to ascertain the dependence on voltage of the passive conductive transport of K and Cl across the human red blood cell membrane. Valinomycin carriers and gramicidin channels are being utilized as model ionophores to compare the transport characteristics of exogenously added known carriers and channels with the endogenous Ca-induced K conductance system. Additional experiments are proposed to utilize voltage-dependent fluorescence changes associated with Ca-induced K conductance as an assay for reconstituting this transport system in vesicles isolated from red cell membranes. Attempts will then be made to incorporate functional vesicles into planar lipid bilayers for direct electrophysiological analysis. Thus, the characterization of the effect of internal Ca on the membrane potential of intact human red blood cells, as determined in the first phase of this project, will be extended to resealed ghost membranes and to isolated membrane vesicles. Further experiments are planned to test the utility of proposed "electrochronic" dyes, and to evaluate and define further the mechanisms by which optical potentiometric indicators report the membrane potential of red cells and the conditions in which they may be used reliably.